Automatic electrophoresis method and apparatus

ABSTRACT

An electrophoresis apparatus for automatically performing medical assays includes an electrophoresis platform which cooperates with a gantry assembly. The electrophoresis platform and the gantry assembly are movable along paths that are perpendicular to each other. An applicator assembly includes pipettes which transfer fluid samples from a specimen tray to an electrophoresis plate mounted on the electrophoresis platform. The electrophoresis platform then moves to a position into the gantry assembly, where electrophoresis is conducted to separate the samples into different fractions. The electrophoresis platform then moves beneath a reagent pouring station where a reagent is applied to make the separated fractions fluoresce under ultraviolet light. The electrophoresis platform is then moved beneath the gantry assembly again, and an air knife in the gantry assembly spreads the reagent. After incubation and drying of the electrophoresis plate, the electrophoresis platform and gantry assembly are moved relative to one another while the electrophoresis plate is read with the aid of ultraviolet lamps and a photomultiplier tube mounted in the gantry assembly. The gain of the photomultiplier tube is automatically adjusted and the data gathered is automatically edited to remove background noise. The edited results can be printed or displayed on a video monitor. Techniques for calibrating the electrophoresis apparatus are also disclosed.

This is a division of application Ser. No. 08/124,502 now U.S. Pat. No.5,460,709 filed Sep. 21, 1993; which is a continuation-in-part ofapplication Ser. No. 08/079,378 filed Jun. 21, 1993, which is nowabandoned.

BACKGROUND OF THE INVENTION

The present invention is directed in general to the field ofelectrophoretic analysis of liquid samples, such as biologicalspecimens. More particularly, the invention is directed to a method andapparatus for automatically conducting electrophoresis with anelectrophoresis plate.

Valuable information can be obtained by an analysis of certainbiological fluids from a patient, such as blood serum, when diagnosingthe patient's illness. Electrophoresis is known to be an effectivetechnique for separating the various components of such fluid forsubsequent analyses using optical densitometry techniques. The physicalphenomenon underlying electrophoretic analysis is that particles whichhave an effective electric charge and which are deposited on a solid orsemi-solid medium are caused to move with respect to the medium by anelectric field applied across the medium. Particles of different typesmove at different rates, so a mixture of different types of particles isseparated into its different components or fractions by electrophoreticanalysis. These separated fractions may then be stained by exposing themto a suitable reagent so that the fractions can be optically detectedusing visible or ultraviolet light.

The electrophoresis process has been performed through a series ofmanual steps for many years. The manual process typically has startedwith the operator preparing an electrophoresis chamber by fillingappropriate cavities of the chamber with buffer solution. Buffersolution is a liquid used in the electrophoresis process to maintain thesurface of the electrophoretic medium in a moist condition and toprovide an electrical interface to a power source applied to the chamberso that an electric field may be applied to the medium. Theelectrophoresis medium is typically a gel substance such as celluloseacetate or agarose that has been coated onto a Mylar (trademark)substrate to form an electrophoresis plate. The liquid sample to beexamined is typically blood serum, but of course may be other liquids.

After the operator has prepared the electrophoresis chamber, he thenapplies consistent volumes of the samples to precise locations on theelectrophoresis medium. The operator then places the medium into theelectrophoresis chamber so that the edges of the medium are immersed intwo buffer cavities at each of its longitudinal ends. Electrophoresis isthen performed using a precise and consistent high voltage applied for aprecise and consistent interval of time across the buffer cavities.

After electrophoresis has been completed, the operator applies a uniformcoating of a staining reagent or stain to the surface of the medium,allowing a precise and consistent interval of time for the reagent andsamples to chemically combine. The staining reagent is a liquid usedafter electrophoresis to chemically combine with the separated fractionsof the fluid samples, causing the fractions to exhibit opticalcharacteristics.

Next, the operator places the electrophoresis medium into atemperature-controlled oven and incubates it using a precise andconsistent temperature and time interval. Incubation is the process ofcontrolling the chemical reaction between the fractions of the liquidsamples and the staining reagent by means of applying heat for a fixedinterval of time.

Next, the operator dries the electrophoresis medium by increasing theoven temperature for a second precise and consistent temperature andtime interval. The drying process stops the reaction between theseparated fractions and the reagent by removing water from the medium.The medium can then be examined using optical densitometry techniques todetermine which fractions were present in the original samples and tofind their relative proportions.

The manual process described above requires careful attention by theoperator in order to provide accurate and reproducible results. It istherefore not surprising that techniques for performing electrophoresisautomatically have been developed. For example, U.S. Pat. Nos. 4,360,418and 4,391,689 to Golias describe an automated electrophoresis andstaining apparatus and method. U.S. Pat. Nos. 4,810,348, 4,890,247,4,909,920, and 4,954,237 tc Sarrine et al also describe an automatedelectrophoresis apparatus and method. An automated applicator assemblywith pipettes for transferring samples to the electrophoresis mediumduring automated analysis is described in U.S. Pat. Nos. 4,827,780 and4,938,080 to Sarrine et al. All of these patents, which are assigned tothe assignee of the present invention, are incorporated herein byreference.

SUMMARY OF THE INVENTION

An object of the invention is to provide an improved method andapparatus for automatically conducting electrophoresis.

Another object of the invention is to provide an electrophoresis methodand apparatus in which an electrophoresis plate is movable in a firstdirection and an optical means for scanning the electrophoresis plate ismovable in an orthogonal second direction.

Another object is to provide an electrophoresis apparatus having an airknife which spreads a liquid reagent across the electrophoresis plate,and which can additionally be used to remove excess water from the platebefore the samples are deposited on it and to-blow hot air against theplate to help dry it after incubation. A related object is to provideair duct valves to isolate the electrophoresis platform from the ambientatmosphere except when air is being blown through the air knife.

Another object of the invention is to provide a method for automaticallyadjusting the anode voltage supplied to a photomultiplier tube in anautomatic electrophoresis apparatus.

Another object is to provide a method for automatically editing datacollected by an automatic electrophoresis apparatus to reduce backgroundnoise.

Another object is to provide a method for chemically avoiding backgroundnoise due to albumin when isoenzymes of creatine kinase are assayedusing an automatic electrophoresis apparatus.

Another object of the invention is to provide an improved method forcalibrating an applicator assembly having pipettes which transfersamples.

Another object is to provide an improved method for calibrating anelectrophoresis apparatus having a platform which moves anelectrophoresis plate along a first path and a gantry assembly whichmoves optical means for scanning the electrophoresis plate along asecond path that is orthogonal to the first path.

Another object of the invention is to provide an improved method forcalibrating temperature sensors and power supplies in an automaticelectrophoresis machine.

In accordance with a first aspect of the invention, an electrophoresisapparatus includes: a first support for an electrophoresis plate whichincludesan electrophoresis medium layer; first means for moving thefirst support along a first linear path; an optical detector; a secondsupport for the optical detector; and second means for moving the secondsupport along a second linear path that passes over the first linearpath and that is substantially perpendicular to the first linear path.

The first support may be an electrophoresis platform having electrodesthat contact the electrophoresis medium layer.

The electrophoresis apparatus may additionally include an applicatorassembly for depositing at least one liquid sample on theelectrophoresis plate, the applicator assembly being disposed above thefirst linear path, and a reagent pouring station which is also disposedabove the first linear path.

The second support may be a gantry assembly on which the opticaldetector is mounted, an air knife additionally being mounted on thegantry assembly. The air knife may be selectively isolated from theambient atmosphere by one or more motor-operated air duct valves. Aheater may be included in the gantry assembly to heat the air blown bythe air knife to help dry the electrophoresis plate after it has beentreated with reagent and incubated.

The gantry assembly may also be provided with a lamp housing forultraviolet lamps which are part of a removable lamp assembly, and anarrangement for releasably latching the lamp assembly to the lamphousing so that the ultraviolet lamps can easily be replaced.

The optical detector may be a photomultiplier tube whose gain isautomatically adjusted before data is gathered by scanning each trackand reducing the anode voltage supplied to the photomultiplier tube eachtime the output of the photomultiplier tube, as indicated by the outputof a photomultiplier tube amplifier, exceeds a predetermined value. Theamplifier may have an adjustable gain and an adjustable offset. The datacollected by the automatic electrophoresis apparatus may be stored inmemory and automatically edited by ignoring peaks that occur outsidepredetermined ranges, and by establishing a base line for peaks withinthe predetermined ranges.

In accordance with a second aspect of the invention, a method forcalibrating an electrophoresis apparatus which has a lamp for emittingultraviolet light, a support for receiving an electrophoresis plate, andan optical detector for scanning the electrophoresis plate while it isexposed to ultraviolet light, includes the steps of: (a) placing acalibration template on the support, the calibration template having afirst fluorescent line and a second fluorescent line that isperpendicular to the first line; (b) clearing a first position counter;(c) clearing a second position counter; (d) actuating a first motor tomove the support and the sensor relative to one another so that thesensor passes over and detects the first line, a first position encoderbeing operatively connected to the first motor, the first positionencoder emitting pulses as the first motor rotates; (e) using the firstposition counter to count the pulses emitted by the first positionencoder while step (d) is conducted; (f) storing the count reached bythe first position counter when the sensor detects the first line; (g)actuating a second motor to move the support and sensor relative to oneanother so that the sensor passes over and detects the second line, asecond position encoder being operatively connected to the second motor,the second position encoder emitting pulses as the second motor rotates;(h) using the second position counter to count the pulses emitted by thesecond position encoder while step (g) is being conducted; and (i)storing the count reached by the second position counter when the sensordetects the second line.

In accordance with a third aspect of the invention, a method forcalibrating an applicator assembly having a first member, a barrel thatis vertically mounted on the first member and that has a bottom end, asecond member, and a plunger that is vertically mounted on the secondmember and that extends into the barrel, includes the steps of: (a)clearing a first position counter; (b) clearing a second positioncounter; (c) actuating a first motor to move the first member to anelevated position above a support, a first position counter beingoperatively connected to the first motor, the first position encoderemitting pulses as the first motor rotates, the pulses emitted by thefirst position encoder being counted by the first position counter; (d)checking the distance between the support and the bottom end of thebarrel with a go/no-go feeler gauge to determine whether the bottom endof the barrel lies within a first predetermined range of distances fromthe support; (e) if the bottom end of the barrel does not lie within thefirst predetermined range of distances from the support, actuating thefirst motor again to move the first member to a different position abovethe support; (f) repeating steps (d) and (e) until the bottom end of thebarrel lies within the first predetermined range of distances from thesupport; (g) storing the count reached by the first position counterwhen the bottom end of the barrel lies within the predetermined range ofdistances from the support; (h) actuating a second motor to move thesecond member to an elevated position above the first member, the secondmotor being fixedly mounted with respect to the first member, a secondposition encoder being operatively connected to the second motor, thesecond position encoder emitting pulses as the second motor rotates, thepulses emitted by the second position encoder being counted by thesecond position counter; (i) checking the distance between the first andsecond members with a go/no-go feeler gauge to determine whether thedistance between the members lies within a second predetermined range;(j) if the distance between the first and second members does not liewithin the second predetermined range, actuating the second motor againto change the distance between the first and second members; (k)repeating steps (i) and (j) until the distance between the first andsecond members lies within the second predetermined range; and (l)storing the count reached by the second position counter when thedistance between the first and second members lies within the secondpredetermined range.

In accordance with a fourth aspect of the invention, a method foranalyzing a liquid sample includes the steps of: (a) depositing thesample on an electrophoresis medium layer; (b) establishing an electricfield across the electrophoresis medium layer; (c) pouring a reagent onthe electrophoresis medium layer; (d) spreading the reagent by blowingair against the electrophoresis medium layer through an air knife slotwhile moving the air knife slot and the electrophoresis medium layerwith respect to one another; (e) shining ultraviolet light on theelectrophoresis medium layer; and (f) scanning the electrophoresismedium layer with an optical sensor.

In accordance with a fifth aspect of the invention, a method forassaying isoenzymes of creatine kinase in a liquid sample includes thesteps of: (a) placing the liquid sample in a receptacle; (b)transferring the sample to an electrophoresis medium layer; (c)establishing an electric field across the electrophoresis medium layer;(d) depositing a reagent on the electrophoresis medium layer; (e)shining ultraviolet light on the electrophoresis medium layer; (f)scanning the electrophoresis medium layer with an optical sensor; and(g) exposing the sample to a Ph indicator dye before step (f) isconducted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view illustrating an electrophoresis apparatusin accordance with the present invention, along with auxiliary devicesused with the apparatus;

FIG. 2 is a perspective view schematically illustrating major componentsinside the housing of the apparatus;

FIG. 3 is a perspective view schematically illustrating air duct systemsinside the housing;

FIG. 4 is a perspective view of an electrophoresis plate that is usedwith the electrophoresis apparatus;

FIG. 5 is a top view of an electrophoresis platform in the apparatus,and additionally shows a sample tray resting on the platform;

FIG. 6 is a side view, partially in section, illustrating theelectrophoresis platform, a transport assembly which moves the platform,and an interlock receptacle which transfers power for electrophoresiswhen the platform is at a withdrawn position;

FIG. 7 is a front view of an air valve in one of the air duct systemsshown in FIG. 3;

FIG. 8 is an exploded perspective view of a reagent applicator assembly;

FIG. 9 is a front view of the gantry assembly;

FIG. 10 is a rear view of the gantry assembly;

FIG. 11 is a bottom view of the gantry assembly;

FIG. 12 is an exploded perspective view, partially broken away,illustrating a lamp assembly which is releasably received in a lamphousing of the gantry assembly;

FIG. 13 schematically illustrates exposure of the electrophoresis plateby ultraviolet lamps in the gantry assembly and measurement of theresulting florescence by a photomultiplier tube in the gantry assembly;

FIG. 14 is a sectional view illustrating air guides provided by thegantry assembly;

FIG. 15 is a rear view of the electrophoresis apparatus with some of thepanels of the housing removed;

FIG. 16 is a top view schematically illustrating how the platformassembly cooperates with the applicator assembly, the reagent pouringstation, and the gantry assembly;

FIG. 17 is a perspective view schematically illustrating the applicatorassembly;

FIGS. 18, 19, and 20 illustrate a block diagram of the electricalcircuitry of the electrophoresis apparatus;

FIGS. 21A-21M illustrate a flow chart for normal operation of theelectrophoresis apparatus;

FIG. 22 is a graph illustrating an example of data collected by theelectrophoresis apparatus before automatic editing;

FIG. 23 is a graph illustrating the edited data, scaled to showinternational units on the vertical axis;

FIG. 24 is a graph illustrating the edited data, scaled so that the mostprevalent isoenzyme is depicted at 100% full scale.

FIGS. 25A-25C illustrate a flow chart for calibrating a temperaturesensor in the electrophoresis apparatus;

FIG. 26 is a top view of a calibration template;

FIGS. 27A-27D illustrate a flow chart for a calibration procedure whichuses the template of FIG. 26;

FIGS. 28A-28D illustrate a flow chart for an applicator calibrationprocedure; and

FIG. 29 is a side view of a go/no-go feeler gauge used during theapplicator calibration procedure.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 illustrates an electrophoresis apparatus 30 in accordance withthe present invention, along with a keyboard 32, video monitor 34, andprinter 36 that are used with apparatus 30. Apparatus 30 has a housing38 with a forward-projecting portion 40 that has a generally U-shapedchannel 42, the channel 42 providing access to the interior of housing38. Housing 38 also includes an air inlet grill 44, and an air outletgrill 46 is provided on portion 40 of housing 38.

FIG. 2 illustrates the major operational components within housing 38.These include an electrophoresis platform 48, an applicator assembly 50with six pipettes 52, a reagent pouring station 54, and a gantryassembly 56. Reagent pouring station 54 is accessible via a hinged cover58 (see FIG. 1) from outside housing 38. Gantry assembly 56 is movablewithin housing 38 in the direction marked by arrow 60. Electrophoresisplatform 48 is movable along channel 42 from a position outside housing38 to a position inside housing 38, as indicated by arrow 62. Platform48 can be positioned beneath applicator assembly 50, reagent pouringstation 54, and gantry assembly 56.

A computer 62, an electrophoresis power supply 64, and additional powersupplies 66 are also mounted in housing 38. Computer 62 is a personalcomputer, such as a Dell (trademark) computer with an Intel (trademark)386 microprocessor and a hard disk. Electrophoresis power supply 64 is abipolar supply, meaning that it supplies potentials that are bothpositive and negative with respect to ground potential.

An air duct conveys air for cooling electrophoresis power supply 64.Another air duct system is provided for supplying air to electrophoresisplatform 48 when it is disposed inside housing 38. A further air ductsystem is provided for supplying air to gantry assembly 56. These airduct systems will be further described with reference to FIG. 3.

In FIG. 3, one air duct 68 has an air entrance 70 at the front ofhousing 38 and extends straight back to air outlet openings (notillustrated in FIG. 3) at the rear of housing 38. Fans 72 are disposednear entrance 70 to force air through duct 68. Electrophoresis powersupply 64 is disposed inside duct 68.

The air duct system for supplying air to gantry assembly 56 has an airinlet portion 74 and an air outlet portion 76. Air inlet portion 74 hasan air entrance 78 disposed adjacent air entrance 70. A fan 80 isdisposed in inlet portion 74, and an air duct valve 82 is provided infront of fan 80 to selectively open or close air inlet portion 74 of theduct. A collar 84 at the inner end of air inlet portion 74 is connectedby a bellows 86 (see FIG. 15) to gantry assembly 56. The inner end ofair outlet portion 76 has a similar collar (not illustrated) which isconnected to a bellows 88 (see FIG. 15) which in turn is connected togantry assembly 56. A fan 90 is provided in air outlet portion 76 and anair duct valve 92 is disposed just outside of fan 90 to selectively openor close portion 76.. Air outlet portion 76 has an air exit 94.

The air duct system for electrophoresis platform 48 includes an airinlet portion 96 and an air outlet portion 98. Portion 96 has an airentrance 100 adjacent air entrance 78 and, at its inner end, has fans102 to direct incoming air to electrophoresis platform 48 when thelatter is in its withdrawn position. The inner end of portion 98 has an,opening (not illustrated) which receives this air. Fans 104 are disposedin portion 98 of the duct, which has an air exit 106 that is positionedadjacent air exit 94. Air exit 106 and 94 are positioned behind airoutlet grill 46 (see FIG. 1). Air entrances 70, 78, and 100 arepositioned behind an air intake filter 108, which is housed behind airinlet grill 44.

FIG. 4 illustrates an electrophoresis plate 110 which is used onelectrophoresis platform 48. Plate 110 includes a substrate 112 made,for example, of a thin Mylar (trademark) plastic sheet. Substrate 112supports an electrophoresis medium layer 114 having a first end portion116, a second end portion 118, and a central portion 120.Electrophoresis medium layer 114 is a stiff gel which includes water anda microporous support medium such as agarose for the water. The term"microporous" means that the electrophoresis medium has tiny pores whichreleasably hold the water, somewhat in the manner of an extremelyfine-grain sponge. A buffer is added to the water to make itelectrically conductive and to adjust its Ph. A surfactant such asmethyl cellulose and other components are preferably included in thewater.

End portion 116 has six holes 122 and, similarly, end portion 118 hassix holes 124. Substrate 112 has an alignment hole 126 and an alignmentslot 128. Although not illustrated in FIG. 4, substrate 112 also has sixholes aligned with the holes 122 in the end portion 116 ofelectrophoresis medium layer 114 and six holes aligned with the holes124 in the end portion 118 of electrophoresis medium layer 114.

From the above description of electrophoresis plate 110 it will beapparent that the term "plate" does not imply a rigid structure;instead, electrophoresis plate 110 is rather flexible. Further detailsabout electrophoresis plate 110 are available in application Ser. No.08/086,918, filed Jul. 7th, 1993 by Philip A. Guadagno et al, thedisclosure of which is incorporated herein by reference.

FIG. 5 illustrates a top view of electrophoresis platform 48. Platform48 includes a plastic tray 130 with a recessed region 132. A pair ofribs 134 extend upward from tray 130 in recessed region 132, and troughs136 are provided in recessed region 132 outside of ribs 134. Tray 130has a central opening 138 in recessed region 132. A heat-transfer member140 is mounted beneath tray 130 and protrudes through opening 138.Member 140 has a top surface that is flush with the surface of tray 130in recessed region 132. A plastic film 142 is adhesively attached totray 130 at the periphery of opening 138 and covers member 140 toprotect it from moisture.

Six electrodes 144 are mounted on tray 130 at one end of opening 138 andsix electrodes 146 are mounted on tray 130 at the other end of opening138. These electrodes are made of compressed graphite. Alignment pegs148 and 150 extend upward from tray 130 in recessed region 132. Holes152 are provided in tray 130 to accommodate screws (not illustrated) formounting tray 130 on underlying components of electrophoresis platform48 (which will be described later). A flexible sealing member 154 ismounted on tray 130 around recessed region 132.

When electrophoresis plate 110 of FIG. 4 is mounted in tray 130,alignment peg 150 extends through hole 126 and alignment peg 148 extendsthrough slot 128. Furthermore, electrodes 146 extend through holes 124and electrodes 144 extend through holes 122.

Tray 130 also has another recessed region, identified by referencenumber 156, for accommodating a removable sample tray 158. Tray 158 hasa first row 160 of sample wells 162 and a second row 164 of sample wells162. As shown, each row has six sample wells 162. Sample tray 158additionally includes a trough 166 for a cleaning solution to wash thepipettes 52 (see FIG. 2, for example) of applicator assembly 50, and atrough 168 for water to wash the cleaning solution from the pipettes.The pipettes 52 transfer liquid samples from one of the row's samplewells 162 to wells 170 (see FIG. 4) molded into electrophoresis mediumlayer 114. A strip of paper (not illustrated) is deposited on region 172of sample tray 158 so that the pipettes 52 can be blotted during thepipette cleaning procedure. Blotting region 172 has six depressions 174which are positioned beneath the blotting the paper to avoid damagingpipettes 52 when they are pressed against the blotting paper.

Turning next to FIG. 6, electrophoresis platform 48 also includes a heatsink 176 having fins 178. Side plates 180 connect heat sink 176 to abottom plate 182. A printed circuit board 184 is connected to the topside of heat sink 176. PCB 184 has a central opening (not numbered) inwhich a pair of Peltier devices 186 are disposed. Peltier devices 186are sandwiched between heat-transfer member 140 and heat sink 176, andcan supply heat to or withdraw heat from heat-transfer member 140 inorder to heat or cool electrophoresis plate 110.

As will be seen from FIG. 6, the electrodes 146 are mounted in blindbores in tray 130 and are connected by screws (not numbered) to a metalstrap 188. Similarly, electrodes 144 are mounted in blind bores and areconnected by screws (not numbered) to a metal scrap 190.

Although not shown in FIG. 6, PCB 184 has a conductor pattern on its topsurface and a conductor pattern on its bottom surface, the conductorpattern on the bottom surface of PCB 184 being electrically insulatedfrom heat sink 176. The conductive patterns on the top and bottomsurfaces of PCB 184 are connected where appropriate by plated-throughholes. Electrical power is supplied to Peltier devices 186 through theconductor patterns. Additionally, a platform temperature sensor(identified by reference number 192 in FIG. 19) is mounted onheat-transfer member 140 and is electrically connected to conductors onPCB 184. A flexible cable (not illustrated) is attached to PCB 184 topermanently connect Peltier devices 186 and temperature sensor 192 toexternal circuitry. However, as a safety feature the electrodes 146 and144 are not permanently connected to electrophoresis power supply 64(FIG. 2).

In FIG. 6, a spring contact 194 is connected to a conductor on the topsurface of PCB 184. A similar spring contact, not numbered due to thesmall scale of the drawing, makes electrical contact with electrodes144. These spring contacts are electrically connected to correspondingconductors at the bottom side of outer end 196 of PCB 184. An interlockreceptacle 198 mounted inside housing 38 (FIG. 1) makes electricalcontact with these conductors and is thus able to provide high voltagefor electrodes 144 and 146 only when platform 48 is in its withdrawnposition.

Further details about electrophoresis platform 48 are explained inapplication Ser. No. 08/079,229, filed Jun. 21st, 1993 by Robert J.Sarrine, the disclosure of which is incorporated herein by reference.

Electrophoresis platform 48 is mounted on a transport assembly 200 whichincludes a base 202 and end members 204 and 206 mounted on base 202. Twoguide bars 208 are fixed to end members 204 and 206, and a shaft 210 isjournalled for rotation on members 204 and 206 and disposed midwaybetween the shafts 208. Shaft 210 is finely threaded along most of itslength. A toothed pulley 212 is connected to the outer end of shaft 210.Bottom plate 182 of electrophoresis platform 48 is mounted on a chassis214 which rides on guide bars 208 and which encloses a nut (notillustrated) that meshes with the threaded portion of shaft 210, so thatrotation of pulley 212 and thus shaft 210 causes chassis 214 to movealong guide bars 208 as indicated by arrow 216. A bellows 218 isconnected between end member 204 and chassis 214 and another bellows,numbered 220, is connected between chassis 214 and end member 206. Thepurpose of bellows 218 and 220 is not to convey air as part of thepreviously-described duct systems, but instead to protect guide bars 208and shaft 210 from dust and debris. Transport assembly 200 iscommercially available from Thompson Industries, Inc. of FortWashington, N.Y., under the trademark "Superslide."

FIG. 7 illustrates air ducts valve 82. It includes a back plate 222 anda front plate 224 that is connected to back plate 222 with screws. Frontplate 224 has a rectangular opening 226 which is aligned with acorresponding, opening in back plate 222. A duct valve motor 230 has aflange 228 that is connected to back plate 222 by screws 232. Motor 230has an internal nut (not illustrated) which engages a threaded shaft234, which is moved linearly when the nut is rotated by motor 230. Thebottom end of shaft 234 is connected to an intermediate plate 235 whichis slidably mounted between plates 222 and 224. It will be apparent thatmotor 230 can move plate 235 into a position where it blocks opening226, or withdraw it from the opening to permit passage of air throughthe duct portion in which valve 82 is mounted. Air duct valve 92 has thesame construction as valve 82.

FIG. 8 illustrates reagent pouring station 54, which includes mountingmembers 236 and 238. A receptacle for a reagent vial 240 includes afirst receptacle portion 242 and a second receptacle portion 244. Aspring finger 246 is mounted on receptacle portions 242 and 244 toretain vial 240 in the receptacle. Receptacle portion 242 has a peg 248which extends into a bore 250 to journal portion 242 for rotation withrespect to mounting member 238. Receptacle portion 244 has a stem 252with a pair of flat cam surfaces 254 (only one of which is shown) on it.One side of mounting member 236 has a recess 256 which receives thefront end of a reagent drive motor 258. Motor 258 is a gear motor (thatis, it includes reduction gearing built into the motor housing). Theshaft 260 of motor 258 is connected to stem 252, extending into anopening 262 in the stem.

Limit switches 262 are attached to mounting member 236 by screws 264(only one of which is shown) and a backing plate 266. Switches 262 arepositioned to engage the cam surfaces 254 to detect whether vial 240 isupside down or right side up. As was indicated previously, an empty vial240 can be withdrawn from reagent pouring station 54 via hinged cover 58(see FIG. 1) and replaced by a full vial of reagent.

Gantry assembly 56 will now be described with reference to FIGS. 9-14.Gantry assembly 56 has a base 268 with an optical window 270 and apneumatic window 272 in it. A brace 274 is mounted on base 268 andextends across window 270, leaving two equal portions 276 and 278 ofwindow 270 unobstructed. A collimator 280 is mounted on brace 274.Collimator 280 is a short, hollow tube which is closed at its upper andlower ends, except for slits 282 in the ends. Slits 282 are aligned, sothat light passing through the lower slit 282 also passes through theupper slit 282 only if the rays are parallel to the longitudinal axis ofcollimator 280.

A lamp housing 284 is mounted on base 268 above optical window 270.Housing 284 has a latch plate 286 (see FIG. 12; latch plate 286 is notshown in FIGS. 9 and 10) to cooperate with a latch 288 (see FIG. 12) ona lamp assembly 290. Lamp assembly 290 includes a support 292, agenerally U-shaped printed circuit board 294 connected to support 292,and a pair of ultraviolet lamps 296 which are connected to the arms 298of PCB 294 by straps 300. Latch 288 is pivotally mounted on protrusions302 extending from support 292 and is biased by a spring (notillustrated) so as to urge the tooth 304 of latch 288 downward.

The walls of lamp housing 284 have grooves 306 which slidably receivethe edges of arms 298 of PCB 294. When lamp assembly 290 is fullyinserted, the tooth 304 of latch 288 slips over the tooth 308 of latchplate 286 to releasably lock lamp assembly 290 inside housing 284. Inthis inserted position, part of one of the lamps 296 is exposed throughunobstructed portion 276 of optical window 270 and part of the otherlamp 296 is exposed through-unobstructed portion 278 of window 270;collimator 280 extends upward between the arms 298 of PCB 294.

A housing 310 for a photomultiplier tube or PMT 312 is mounted on lamphousing 284. A socket 313 for PMT 312 is mounted on housing 310.Reference number 316 identifies a mirror which is mounted on a plate 314which in turn is mounted on an extending portion 315 of housing 284.Mirror 316 is positioned at an opening (not illustrated) in the side ofPMT housing 310. Collimator 280 extends through lamp housing 284 andinto PMT housing 310, and mirror 316 reflects light that has passedthrough collimator 280 to PMT 312. Reference number 317identifies anultraviolet filter.

A first air guide 318 is supported over base 268 by braces 319 and 320.First air guide 318 has a collar 322 for connection to bellows 86 (seeFIG. 15). A second air guide 324 is mounted on base 268 over pneumaticwindow 272. Second air guide 324 is open at the bottom, so thatpneumatic opening 272 in base 268 provides an entrance into guide 324.Guide 324 has a collar 326 for connection to bellows 88 (see FIG. 15).

An air knife guide 328 is mounted on guide 324 by screws 330 and issealingly connected to guide 318 by duct tape 332. Air knife guide 328includes a wall 334 which is spaced slightly apart from a wall 336 toprovide an air knife slot 338. An air knife or gantry blower 340 and aheater 342 are connected to a mounting member.344 that is attached toguide 338. Reference number 339 designates a temperature sensor. As willbe seen from FIG. 14, blower 340 extends into the space inside first airguide 318. When blower 340 is turned on, it forces air toward air knifeslot 338, which is positioned at one edge of pneumatic opening 272. Thisair can then be collected via pneumatic opening 272 and air guide 324.

A brace 348 is screwed to first and second air guides 318 and 324 toincrease the structural rigidity of gantry assembly 56 and to provide asupport for mounting a printed circuit board (not illustrated) with someof the electrical circuitry of electrophoresis apparatus 30. Slidebearings 350 are mounted on base 268 along the opposite sides thereof.

Turning next to FIG. 15, which shows the back side of electrophoresisapparatus 30 with some of the outer panels of housing 38 removed, a pairof guide bars 352 (only one of which is shown) are mounted on housing38. These guide bars extend through bores (not illustrated) in slidebearings 350 to mount gantry assembly 56 for lateral movement. A bracket354 is connected to housing 38 and another bracket 356 is connected to asupport 358 attached to air duct outlet portion 76. A toothed pulley 360is rotatably mounted on bracket 356. A toothed pulley 362 is rotatablymounted on bracket 354. Pulley 362 is driven by a gantry drive motor 362which is connected to housing 38. Motor 362 is a stepper motor, withreduction gearing, and furthermore has a rotary position encoder 366(see FIG. 20) attached to the motor housing. A toothed belt 368 isstretched between pulleys 360 and 362 and is connected to gantryassembly 56, so that motor 362 can slide gantry assembly 56 back andforth along guide rods 352 via belt 368.

A platform drive motor 370 is mounted on housing 38. It, too, is astepper motor, and has a rotary position encoder 372 (see FIG. 20)attached to its housing. A toothed pulley 374 is connected to the shaftof motor 370. A toothed belt 376 extends between pulleys 374 and 212. Itwill be apparent that motor 370 moves electrophoresis platform 48forward and backward via belt 376 and transport assembly 200.

FIG. 15 also illustrates a rear panel 378 of housing 38. It includesvarious grills 380 for passage of air and a further air vent 382 that isaligned with a cooling fan (not illustrated) that is part of computer62. Panel 378 also includes a window 384 which exposes variousconnectors 386 at the rear of computer 62.

How electrophoresis platform 48 cooperates with gantry assembly 56 willnow be further explained with reference to FIG. 16. FIG. 16 illustratesa top view of these components, with platform 48 positioned at the frontand gantry assembly 56 positioned at the right as in FIG. 2. Gantryassembly 56 is shown with dotted lines since it is the base 268 thereofthat is depicted. During electrophoresis different fractions of samplesthat have been deposited in wells 170 of electrophoresis plate 110 movephysically at different rates along six tracks that are schematicallyillustrated by dot-dash chain lines 388.

From the foregoing discussion it will be apparent that platform 48 ismovable along a platform path 390. By moving it along this path,platform 48 can be positioned beneath applicator assembly 50, reagentpouring station 54, or gantry assembly 56. Reference number 392schematically illustrates a home switch at the inner end of platformpath 390. Gantry assembly 56 is movable along a gantry path 394 that isperpendicular to platform path 390. Reference number 396 schematicallyillustrates a home switch at the left end of gantry path 394.

During electrophoresis itself, gantry assembly 56 is positioned as shownin FIG. 16 and electrophoresis platform 48 is moved along platform path390 to an electrophoresis position. In this position, electrophoresisplate 110 is disposed directly under pneumatic window 272, and sealingmember 154 engages the underside of base 268 around the periphery ofwindow 272, so that platform 48 and gantry assembly 56 togetherconstitute an electrophoresis chamber. After electrophoresis has beenconducted, platform 48 is moved forward along platform path 390 to aposition beneath reagent pouring station 54, where a vial of reagent ispoured onto plate 110. Platform 48 is then moved back along platformpath 390 to a reagent spread position, which is the same as theelectrophoresis position. Gantry assembly 56 is moved back and forthalong gantry path 394 while air is blown downward gently through airknife slot 338. This spreads the reagent uniformly acrosselectrophoresis medium layer 114. The air received through air knifeslot 338 is exhausted via pneumatic window 272. The reagent can later beremoved by blowing air rapidly through air knife slot 338 while movinggantry assembly 56 across electrophoresis plate 110. This sweeps thereagent into troughs 136.

After incubation and drying of the reagent, which will be described inmore detail subsequently, gantry assembly 56 again cooperates withplatform 48 to obtain optical data for analysis using densitometrytechniques. With platform 48 at the inner position, gantry assembly 56is moved along gantry path 394 until slit 282 is aligned with a firstone of the tracks 388. Ultraviolet light from lamps 296 causes thereagent-treated sample along this track to fluoresce, and fluorescentlight that is emitted directly upward passes through collimator 280 andis reflected by mirror 316 to PMT 312. This is shown schematically inFIG. 13, where ultraviolet filter 317 absorbs any ultraviolet light thatmay be reflected from plate 110. Platform 48 is moved along platformpath 390 to move the first track 388 with respect to slit 282, afterwhich the position of gantry assembly along gantry path 394 is shiftedslightly to bring slit 282 over the second path 388. In this way theelectrophoresis platform 48 and gantry assembly 56 cooperate to scan thesix tracks 388 one by one.

FIG. 17 depicts a schematic illustration of applicator assembly 50 todemonstrate its operational features. Applicator assembly 50 includes aback plate 400 that is mounted (by means not shown) for up and downmovement, as indicated by arrow 402. A pipette barrels motor 406 isconnected to the rear side of plate 400. Motor 406 is a gear motorhaving a positioning encoder 408 (see FIG. 20) built into its housing.Motor 406 has a shaft 410 which protrudes from both ends of the motor. Apinion 412 is connected to each end of shaft 410 and meshes with arespective rack of teeth 414 that is connected to housing 38 of theelectrophoresis apparatus. It will be apparent that motor 406 can beactuated to move plate 400 in the direction of arrow 402. Referencenumber 416 schematically designates a home switch which is closed whenmotor 406 raises plate 400 to a predetermined elevated position.

A pipette bar 418 is connected to a spacer 420 which in turn isconnected to back plate 400. Six pipette barrels 422 are attached topipette bar 418 at the bottom side thereof.

An actuator yoke 424 is mounted (by means not shown) on back plate 400for movement up and down, as indicated by arrow 426. A pair of legs 428extend backward from yoke 424 and terminate in racks of teeth 429. Apipette plungers motor 430 is attached to plate 400 and has a shaft 432to which a pair of pinions 434 are connected. Motor 430 is a gear motorwith an encoder 431 (see FIG. 20) built into its housing. Pinions 434mesh with racks of teeth 429, so that yoke 424 can be moved with respectto back plate 400 in the direction of arrow 426 by actuation of motor430 in the appropriate direction. A home switch that is connected toback plate 400 is schematically illustrated at 436. Switch 436 is closedwhen motor 430 has raised yoke 424 to a predetermined position abovespacer 420.

A plunger bar 438 is connected to the front side of actuator yoke 424.Six pipette plungers 440 are connected to the bottom side of bar 438 andextend through openings 442 in pipette bar 418 and into pipette barrels422, Each barrel 422 and its associated plunger 440 cooperate to form apipette 52, The vertical position of the pipettes 52 is controlled bymotor 406, and motor 430 controls the drawing of fluid into the pipettes52 or the expelling of fluid from the pipettes.

The electrical circuitry of electrophoresis apparatus 30 will now bedescribed with reference to FIGS. 18-20.

Computer 62 includes, inter alia, a CPU 500, a read/write memory 502,,and non-volatile memory in the form of hard disk 504. Disk 504 storesprograms for operating the electrophoresis apparatus 30 and also storesvalues needed such operation, such as calibration values. Computer 62 isconnected to a digital I/O circuit 506 by a bus 508. Computer 62 is alsoconnected to an analog I/O circuit 510 by a bus 512. Analog I/O circuit510 includes D/A and A/D converters (not illustrated) so that circuit510 can receive digital values from computer 62 and supply correspondinganalog voltages to the analog circuitry connected to circuit 510, and sothat it can receive analog voltages from this circuitry and convey thedigital equivalents to computer 62.

Power supplies 66 (see FIG. 2) include a lamp power supply 513, aPeltier power supply 514, and a power supply 516 for photomultipliertube 312. The PMT power supply 516 receives a voltage control signalfrom analog I/O circuit 510 and supplies a PMT voltage designated bythis control signal to the anode (not shown) of PMT 312. A PMT voltagemonitor 518 is connected to power supply 516 in order to supply amonitor signal to circuit 510. This monitor signal is proportional tothe actual output voltage of power supply 516. The output of PMT 312 isamplified by an amplifier 520 and supplied to circuit 510, whichtransfers the amplified PMT output to computer 62 in digital form.Amplifier 520 has a gain input port and an offset input port whichrespectively receive signals from I/O circuit 510 to set the gain orsignal multiplication factor of amplifier 520 and to set the offset orDC level of amplifier 520.

Peltier power supply 514 supplies current, in either a heating directionor cooling direction, to Peltier devices 186. A current monitor 522 isconnected to power supply 514 to provide circuit 510 with a monitorsignal proportional to the actual current output and polarity. As waspreviously mentioned, platform temperature sensor 192 is mounted onheat-transfer member 140 (see FIG. 6) and thus effectively senses thetemperature of Peltier devices 186. Sensor 192 supplies a sensor signalto circuit 510.

Electrophoresis power supply 64 is a bipolar power supply. It has twooutput ports 524 and 526, one of which is positive with respect toground and the other of which is negative with respect to ground.Circuit 510 supplies power supply 64 with a control signal which servesto set the potential at the positive port to a designated value between0 and 750 volts and to set the potential at the negative port to minusthe designated value. Ports 524 and 526 are connected to electrodes 144and 146 by interlock receptacle 198 (see FIG. 6) when electrophoresisplatform 48 is at the electrophoresis position. An electrode current andvoltage monitor 528 is connected to power supply 64 to supply monitorsignals to circuit 510.

A gantry heater control circuit 530 receives a control signal fromcircuit 510 and drives gantry heater 342 at a power level determined bythe control signal. Gantry temperature sensor 443 supplies circuit 510with a sensor signal.

Air knife or gantry blower 340 (see FIG. 14) includes a gantry blowermotor 532 that is driven by a motor control circuit 534 which receives acontrol signal from circuit 510.

Fans 102 and 104 (see FIG. 3) include duct fan motors 536 and fans 80and 90 (FIG. 3) include duct fan motors 538. Motor control circuits 540and 542 receive signals from circuit 506 to turn these fans on or off. Amotor control circuit 544 connected to I/O circuit 506 controls ductvalve motors 230 to open or close air duct valves 82 and 92 (see FIG.3). Similarly, a motor control circuit 546 receives a control signalfrom circuit 506 and drives gantry drive motor 364 accordingly; a motorcontrol circuit 548 receives a control signal from circuit 506 anddrives pipette plungers motor 430 accordingly; a motor control circuit550 receives a control signal from circuit 506 and drives pipettebarrels motor 406 accordingly; a motor control circuit 552 receives acontrol signal from circuit 506 and drives reagent drive motoraccordingly; and a motor control circuit 554 receives a control signalfrom circuit 506 and drives platform drive motor 370 accordingly.Position encoders 366, 372,408, and 431 emit pulses to circuit 506 asthe respective motors rotate, each pulse indicating that the respectivemotor has rotated through a small predetermined angle.

Home switch 392 emits a signal to circuit 506 when electrophoresisplatform 48 is located at its home position (see FIG. 16, for example).Home switch 396 emits a signal to circuit 506 when gantry assembly 56 isat its home position. Similarly, home switches 416 and 436 emit signalsto circuit 506 when the pipette barrels motor 406 and pipette plungersmotor 430 are at their home positions.

Hard disk 504 stores a program for operating electrophoresis apparatus30 during normal operation to perform an assay. It also stores varioususer-programmable values, such as temperatures and times to be usedduring assays, along with user-programmable options such as how theresults of assays are to be presented. Hard disk 504 additionally storesvalues which characterize various components of apparatus 30 and whichare used by the program when apparatus 30 is employed to conduct anassay. For example, the characteristics of the temperature sensors andthe summed values of encoder pulses which represent particular positionsof mechanical components are stored beforehand for use by the program.Approximately-correct default values are stored when apparatus 30 ismanufactured but it is preferable to calibrate apparatus 30 before useto replace these default values with improved values that arecharacteristic of the individual apparatus 30. However, a discussion ofthe calibration procedures will be delayed until normal operation ofapparatus 30 to conduct an assay is described with reference to theprogram illustrated in FIGS. 21A-21M.

This program will be described in the context of a typical use forelectrophoresis apparatus 30, which is to assay the isoforms of creatinekinase of a patient to confirm a diagnoses of myocardial infarction.These isoforms include the MM isoenzyme or fraction (which is associatedwith muscular exercise or injury or a muscle-wasting disease), the MBisoenzyme or fraction (which is associated with heart tissue), and theBB isoenzyme or fraction (which is associated with the nervous anddigestive systems). Measurements of the actual and relative quantitiesof these isoenzymes, particularly at different times to indicate trends,provide physicians with valuable diagnostic information.

In a typical situation blood would be drawn from a patient three timesat hourly intervals and centrifuged to provide three plasma samples. Theoperator conducting the assay would place these three samples in threeof the wells 162 (see FIG. 5) in one of the rows 160 or 164 of sampletray 158. The operator would place a normal control fluid, an abnormalcontrol fluid, and a reference/calibrator fluid in the remaining threewells 162 of the row. The operator would then place the sample tray 158and an electrophoresis plate 110 on electrophoresis platform 48 beforeturning electrophoresis apparatus 30 on.

With reference to FIG. 21A, ultraviolet lamps 296 and photomultipliertube 312 are turned on in step 600. The lamps and PMT need to warm upbefore they stabilize, so a warm-up timer is set to two minutes. Next,at step 602, the position of gantry assembly 56 is ascertained. If it ispositioned at home switch 396 (see FIG. 16), a gantry position counteris cleared in step 604. If gantry assembly 56 is not located at switch396 when step 602 is conducted, it is moved to that position in step 606before the gantry position counter is cleared. The gantry positioncounter is an up/down counter which counts pulses from the positionencoder 366 (see FIG. 20), counting up when gantry assembly 56 movesaway from home switch 396 and counting down when it moves toward homeswitch 396, so after step 604 has been completed the content of thegantry position counter continuously corresponds to the position ofgantry assembly 56 along gantry path 394. Gantry assembly 56 is moved inthe right direction, to the position shown in FIG. 16, during step 608.This is done by using motor control circuit 546 (see FIG. 20) to drivemotor 364 in the desired direction until the contents of the gantryposition counter are equal to a previously-stored count valuecorresponding to the gantry position shown in FIG. 16.

Similarly, the position of electrophoresis platform 48 is ascertained insteps 610, and if it is not already located at home switch 392 (FIG. 16)it is moved there in step 612 before a platform position counter iscleared in 614. Platform 48 is moved to the front position shown in FIG.16 in step 616. Similarly, the position of plungers 440 (see FIG. 17) ischecked in step 618, and if they are not at their top position they aremoved to that position in step 620 before a plunger position counter iscleared in step 622. Like the remaining position counters to bedescribed below, the plunger position counter and the platform positioncounter are up/down counters. In step 626 the position of pipette 52(or, more accurately, barrels 422) is checked, and if they are not attheir top position already they are moved there in step 628, after whicha pipette position counter is cleared in step 630. Duct valve motors 230(see FIGS. 7 and 20) are stepping motors which are over-driven in step632 to ensure that duct valves 82 and 92 are at the closed position,regardless of their positions before step 632 was conducted. After step632 has been completed, duct valves 82 and 92 can be opened or closedsimply by driving motors 230 to move their shafts 234 a predetermineddistance in the opened or closed direction.

Pipettes 52 are washed in step 640. With reference to FIGS. 2 and 16,this is done by moving electrophoresis platform 48 so that the trough166 of washing solution is beneath pipettes 52, lowering the pipettesinto the washing solution, reciprocating plungers 440 several timeswhile the pipettes are immersed, raising the pipettes above trough 166and lowering the plungers to expel any remaining washing solution,moving platform 48 until water trough 168 lies beneath pipettes 52,lowering the pipettes again and reciprocating the plungers severaltimes, raising the pipettes and lowering the plungers to expel anyremaining water, moving platform 48 until blotting region 172 liesbeneath pipettes 52, lowering the pipettes to blot them against a stripof paper (not illustrated) deposited on region 172, and then raising thepipettes again.

In step 642 samples are transferred from a row of wells 162 on sampletray 158 to the corresponding wells 170 of electrophoresis plate 110(see FIG. 4). Step 642 is conducted by moving electrophoresis platform48 until the row of wells 162 lies beneath pipettes 52. The pipettes arethen lowered into the wells. Plungers 440 are then raised to draw onemicroliter of fluid into each pipette, and the pipettes are then raised.Platform 48 is then moved so that pipettes 52 are above blotting region172, whereupon the plungers are lowered to expel the samples onto theblotting paper. Platform 48 is then moved slightly so that a freshregion of blotting paper lies beneath pipettes 52, which are thenblotted against the-paper. After the pipettes are raised, platform 48 ismoved until the rows 162 again lie beneath pipettes 52. The pipettes arelowered into the wells and the plungers are raised to draw fivemicroliters of fluid into each pipette. While the pipettes are stillimmersed, the plungers are lowered to expel the samples back into wells162. This agitates the samples and removes any bubbles that maypreviously have been present. Then the plungers are raised to draw twomicroliters into each pipette. The pipettes themselves are then raisedand one microliter is expelled back into the sample wells, leaving onemicroliter in each pipettes. Drawing in two microliters and expellingone helps to avoid bubbles at the lower ends of the pipettes. At thispoint the platform 48 is moved again until the wells 170 ofelectrophoresis plate 110 lie beneath pipettes 52. The plungers 440 arelowered first, so that a drop is formed on the end of each barrel 422,and then the barrels 442 are lowered so that the drops are placed in thesample wells 170. Precisely one microliter of fluid is transferred toeach well 170.

It takes a certain amount of time for the samples in the wells 170 todiffuse into the electrophoresis medium layer 114. To this end, anabsorption timer is set to a user-programmed value (a typical valuewould be a minute and a half) in step 644. The absorption time and otheruser-programmable values which will be discussed later are storedbeforehand, replacing default values that were stored when apparatus 30was manufactured. The pipette washing procedure is executed again instep 646 and thereafter electrophoresis platform 48 is moved to theelectrophoresis position. As was mentioned before in conjunction withFIG. 16, platform 48 is moved to its electrophoresis position by movingit to the rear along platform path 390 until electrophoresis mediumlayer 114 lies directly beneath opening 272 in gantry assembly 56 whenthe gantry assembly is positioned at the right as shown in FIG. 16.

Fans 102 and 104 (see FIG. 3) are turned on in step 650. When platform48 is in the electrophoresis position, heat sink fins 178 (see FIG. 6)are positioned in front of fans 102, and the air blown through fins 178by fans 102 is collected by air outlet portion 98 of the air duct systemand subsequently expel through air exit 106. Additionally, in step 650the Peltier devices 186 (see FIG. 6) are turned on, with the polarity ofthe current supplied to the Peltier devices being such that their bottomsurfaces are heated and PG,44 their top surfaces are cooled. As aresult, heat-transfer member 140 (see FIG. 6) begins to withdraw heatfrom electrophoresis plate 110.

In step 652, a check is made to determine whether the absorption timeset by the absorption timer has expired. After expiration of this perioda further check is made, at step 654, to determine whether platformtemperature sensor 192 (see FIG. 19) has reached the temperature forconducting electrophoresis. Then electrophoresis power supply 64 isturned on in step 656 and an electrophoresis timer is set in step 658. Atypical value for the voltage applied across electrodes 144 and 146 (seeFIG. 5) would be 1500 volts, with a current of 30 milliamps. A typicalelectrophoresis time would be five minutes. During the electrophoresisoperation the 45 watts dissipated in electrophoresis medium 114 istransferred by heat-transfer member 140 (see FIG. 6) and Peltier devices186 to heat sink 176, and the air current through air duct inlet andoutlet portions 96 and 98 (see FIG. 3) carries this heat away. As aresult the electrophoresis medium layer 114 remains at theelectrophoresis temperature despite the current flowing through it.Although not illustrated in the flow chart, monitor 528 (see FIG. 19)continuously monitors the electrophoresis voltage and current throughoutthe electrophoresis procedure.

After the electrophoresis time has expired (step 660), electrophoresispower supply 64, fans 102 and 104, and Peltier devices 186 are turnedoff (step 662). In step 664 the electrophoresis platform 48 is movedforward to the reagent application position beneath reagent pouringstation 54. Reagent drive motor 258 (FIG. 8) is then actuated to invertvial 240 (step 666). Platform 48 is then moved to the rear alongplatform path 390 until it reaches the reagent spread position (step668), which is the same as the electrophoresis position. Duct valves 82and 92 (see FIG. 3) are then opened (step 670) and air knife blower 340(see FIG. 14) is turned on to a slow speed (step 672). Air is drawnthrough air inlet portion 74 (FIG. 3), bellows 86 (FIG. 15), and intoair guide 316 (FIG. 14) of gantry assembly 56. This air is directedagainst electrophoresis plate 110 through air knife slot 338 (FIG. 14)and is then removed via air guide 324 of gantry assembly 56, bellows 88(FIG. 15), and air duct outlet portion 76 (FIG. 3). Gantry assembly 56is moved back and forth four times (step 674) to permit the air knife tospread the reagent over portion 120 (see FIG. 4) of electrophoresismedium layer 114. Then air knife blower 340 is turned off (step 676),duct valves 82 and 92 are closed again to pneumatically isolate gantryassembly 56 from the external atmosphere (step 678), and a reagentabsorption timer is started to time a two minute period for the reagentto be absorbed (step 680).

After the two minute absorption period has expired (step 682), ductvalves 82 and 92 are opened again (step 684). In step 686 gantryassembly 56 is moved to a spread start position. With reference to FIG.16, in the spread start position air knife slot 338 lies to the right ofelectrophoresis medium layer 114. Air knife blower 340 is then turned onat a high speed in step 688 and gantry assembly 56 is moved to a spreadend position, in which air knife slot 338 is positioned to the left ofelectrophoresis medium layer 114. Thus the air knife makes one sweepacross electrophoresis medium layer 114 and the air blows at arelatively high speed to remove the reagent remaining on electrophoresismedium layer 114. Blower 340 is turned off in step 700. During operationof the air knife (steps 672-676 and, particularly steps 686-700) thereagent blown from electrophoresis plate 110 accumulates in troughs 136(see, for example, FIG. 16). Duct valves 82 and 92 are closed in step702.

Electrophoresis plate 110 must then be incubated while the reagentchemically combines with the isoenzymes that have been separated by theelectrophoresis procedure. Gantry assembly 56 is moved to an incubationposition (which is the same as the electrophoresis position) in step704. Peltier devices 186 are turned on in step 706, with the polarity ofthe current being such that electrophoresis plate 110 is heated.Furthermore, fans 102 and 104 are turned on to blow air across the heatsink fins 178 (see FIG. 6). In step 708 a check is made to determinewhether electrophoresis platform 48 (or, more accurately, platformtemperature sensor 192 as shown in FIG. 19) is up to the incubationtemperature of 45° C. An incubation timer is set in steps 710 after thetemperature reaches a user-programmed value (such as 45° C.). The timeset by the incubation timer is also user-programmable (a typical timewould be five minutes).

Duct valves 82 and 92 are opened in step 714 after the incubation periodhas expired. In a matter analogous to the fixing step duringphotographic processing, the electrophoresis medium layer 114 must nowbe dried to halt the chemical reaction between the reagent and theisoenzymes. In step 716 the heating current to Peltier devices 186 isincreased and, furthermore, heater 342 (see FIG. 14) is turned on. Airknife blower 340 is also turned on. Furthermore fans 102 and 104 areturned on to blow air across heat sink fins 178. Gantry assembly 56 ismoved slowly back and forth across electrophoresis plate 110 during step718.

The drying temperature and the drying time are user-programmable.Typical values would be 54° C. and two minutes, respectively. In step720 a check is made to determine whether a drying timers has beenstarted. If not, a check is made in step 722 to determine whetherplatform temperature sensor 192 (see FIG. 19) and gantry temperaturesensor.443 have reached the drying temperature. The drying timer isstarted in step 724 after the drying temperature has been reached.

Returning to step 720, after the drying timer has been started a checkis made at step 726 to determine whether it has timed out. Peltierdevices 186, gantry heater 342, air knife blower 340, and fans 102 and104 are turned off after the drying time has expired, and duct valves 82and 92 are closed again (step 728 and 730).

The voltage supplied to the anode of photomultiplier tube 312 must beset before PMT 312 is used to collect data from electrophoresis plate110. The gain of a PMT is a function of the anode voltage. The generalequation for the gain G is set forth in Equation 1:

    G=kV.sup.αn                                          (1)

In Equation 1, V represents the anode voltage, n represents the numberof stages in the photomultiplier tube, and k and α are constants whichare available from the manufacturer of the tube. The PMT 312 ispreferably a nine stage tube (n=9) available from Hamamatsu PhotonicsK.K. of Japan, with a US sales office at 360 Foothill Road, P.O. Box6910, Bridgewater, N.J. 08807.

As was mentioned earlier the gain of PMT amplifier 520 (see FIG. 19) isadjustable. However it is desirable PMT tube 312 itself to contribute asubstantial portion of the overall gain in order to achieve a goodsignal to noise ratio.

Although not illustrated in FIG. 19, analog I/O circuit 510 includes anA/D converter which receives the output of amplifier 520. This A/Dconverter is capable of converting analog signals in the minus fivevolts to plus five volts range to twelve bit digital signals, plus asign bit. That is, the A/D converter is capable of dividing an inputsignal into 1.22 millivolt segments, with 2¹² (=4096) such segmentsbeing available. A malfunction would occur if the absolute value of theoutput signal from amplifier 520 to the A/D converter exceeded fivevolts, which will hereafter be call the "full scale" value. Plus fivevolts will be called the "positive" full scale value.

In accordance with the present invention, the anode voltage of PMT 312is initially set at a relatively high value to obtain a relatively highgain. Then the six tracks 388 (see FIG. 16) are scanned sequentially.Each time the measured value (that is, the output of amplifier 520)exceeds some predetermined fraction of the full scale value, a new gainis calculated and a reduced voltage is applied to PMT 312 to achieve areduced gain. The new gain is found by dividing the measured value intoa reduction factor, expressed as a fraction of full scale, and bymultiplying the quotient by the old gain. This is shown in Equation 2.

    G.sub.new =G.sub.old ×R/M                            (2)

In Equation 2, R represents the reduction factor and M represents themeasured value. In electrophoresis apparatus 30, M has been selected tobe one-half of the positive full scale value, or 2.5 volts, and R hasbeen selected to be one-fourth of the positive full scale value, or 1.25volts. Accordingly, each time the measured value exceeds 2.5 volts, anew gain which does not exceed one-half the old gain is calculated, withthe exact value of the new gain depending upon the measured value.

The voltage that needs to be applied to the anode of PMT 312 to achievethe desired gain can be determined by solving Equation 1. This voltageis shown in Equation 3. ##EQU1## In Equation 3, the gain value G is, ofcourse, the new gain.

In FIG. 21H, Peltier devices 186 are turned on to cool electrophoresisplate 110 in step 732. In step 734 a check is made to determine whetherplatform temperature sensor 192 (FIG. 19) is down to the scantemperature (200° C.). When it has reached the scan temperature, afurther check is made at step 736 to determine whether the warm-up timerthat was set in step 600 has timed out. Then the gain of PMT amplifier520 is set to one and the offset is set to zero in step 738.Additionally, the anode voltage is set at 647 volts for an initial PMTgain of approximately 400 with the tube that is used. In step 740 atrack counter is set to one. Track one is the right-most track 388 shownin FIG. 16. Track six is the left-most track 388.

Gantry assembly 56 is moved so that slit 282 is aligned with track onein step 742. In step 744 electrophoresis platform 48 is moved to thescan start position. In the scan start position slit 282 is locatedbefore the tracks begin, below wells 170 as depicted in FIG. 16.Platform 48 begins moving toward the front of apparatus 30 in step 746to begin scanning track one. The reagent that is bound to the isoformsalong track one fluoresces under the influence of ultraviolet lamps 296(see FIG. 13, for example), and collimator 280 (see FIG. 11) permitsfluorescent light that is emitted directly beneath it in a directionperpendicular to electrophoresis plate 110 to reach PMT 312. The outputof PMT 312 is amplified by PMT amplifier 520, the output of which ismonitored during the scan. If the output of amplifier 520 exceeds 2.5volts (that is, half of the positive full scale value), the anodevoltage on PMT 312 is reduced (as previously discussed) in step 750 toreduce the PMT gain. A check is made, at step 752, to determine whetherplatform 48 has reached the track end position. In FIG. 16, the trackend position is located above the dot-dash chain lines that are used todepict tracks 388. After it reaches the track end position, platform 48returns at a relatively high speed to the track start position (step754) and the track counter is incremented. In step 756 a check is madeto determine whether the track number exceeds six. If not, furthertracks remain to be scanned and processing returns to step 742. Thebrightest point on plate 110 will lead to an amplifier output between1.25 and 2.5 volts after all six tracks have been scanned.

After the anode voltage for the PMT has been set, the gain and offset ofPMT amplifier 520 are set on a track-by-track basis, with adata-gathering run being made over each track after the gain and offsethave been set. To do this, the track counter is set again to one in step758, and gantry assembly 56 is moved again to the track one position instep 760. Platform 48 is moved to the scan-start position in step 762.Initial values for a low register and a high register are set in steps764 and 766 and platform 48 begins moving toward the front of apparatus30 in step 768 to begin scanning track one. A check is made in step 770to determine whether the present or current output of PMT amplifier 520is greater than the value stored in the low register; if so, the valuestored in the low register is replaced by the present or current outputof amplifier 520 in step 772. A check is then made in step 774 todetermine whether the present or current output of amplifier 520 ishigher than the value stored in the high register, and if so the oldvalue is replaced by the present value in step 776. The high and lowvalues detected during the scan are stored after platform 48 has reachedits end position (step 778 and 780). Then the offset of amplifier 520 isset so that the amplifier output is zero at the lowest point detectedduring the scan and the amplifier gain is set so that the highest pointdetected during the scan leads to an output of 4.5 volts (steps 782 and784). Platform 48 is returned to the scan start position in step 786.Then a data collection scan is made in step 778, and the data is stored.The track counter is incremented at step 790, and a check is made atstep 792 to determine whether the last track has been read. If not,processing returns to step 760. Peltier devices 186 and fans 102 and 104are turned off (step 794) after the last track has been read, andplatform 48 is returned to its final position at the front of apparatus30 (step 796).

How the results of the measurements are to be presented is auser-programmable option. The testing facility at which apparatus 30 isemployed can elect to have the results automatically scaled or to havethem graphically expressed in international units. This election is madebeforehand, and if automatic scaling is elected the user also selectsthe number of international units that are to represent full scale.During an assay, a check is made at step 800 to determine if the resultsare to be depicted in international units. If so, all six stored scansare scaled in step 810 relative to the selected full-scale value. If theresults are to be automatically scaled ("no" in step 800), the storedscans are all scaled relative to the largest peak in step 812. Afterscaling (steps 810 or 812), the stored values are edited in step 814 toremove background noise and unwanted signals.

Step 814 will be described in more detail with reference to FIG. 22, agraph showing an example of a scan of one track. In FIG. 22, theordinate axis represents the intensity of the light detected byphotomultiplier tube 312 and the abscissa axis represents the distancealong the relevant track 388. The graph shown represents a scan scaledrelative to the largest peak (step 812). Spike 816 represents the MMisoenzyme of creatine kinase. Spike 818 represents the MB isoenzyme, andspike 820 represents the BB isoenzyme.

There are three main sources of background noise. One is airborne lint.Many laundry detergents employ fluorescent materials as brighteners, soan errant fiber from clothing may produce a spurious signal if ithappens to fall on one of the tracks 388. Albumin is another potentialsource of background noise. It is normally present in blood serum orplasma but ordinarily does not cause problems during an assay of theisoenzymes of creatine kinase. The reason is that normal albumin doesnot chemically combine with the reagent used in such an assay. However amodified form of albumin which is naturally fluorescent may be presentin the blood of kidney patients or patients taking an anti-clottingdrug.

The third potential major source of background noise is macro creatinekinase, which is also naturally fluorescent under ultraviolet light.Macro creatine kinase results when certain antibodies bind to creatinekinase, as occasionally happens in elderly patients with certainauto-immune disorders.

The first order of defense employed by electrophoresis apparatus 30against background noise arising from airborne contaminants isavoidance. Duct valves 82 and 92 (see FIG. 3) mechanically isolateelectrophoresis plate 110 from the ambient atmosphere during majorportions of the analytic procedure described above. The risk ofcontamination is reduced accordingly. The duct valves are open only whenthis is necessary for operation of air knife blower 340 (see FIG. 14).

Even if electrophoresis plate 110 does become contaminated with lint,perhaps when the operator installed plate 110 in apparatus 30 or duringthe initial portion of the automatic operation of apparatus 30 beforeelectrophoresis platform 48 is withdrawn into the machine, it isfrequently possible to remove the resulting background noiseelectronically. Arrows 822-830 have been added to FIG. 22 to mark theminima of the curve. These minima are identified by detecting where theslope of the curve changes from negative to positive. Since spikes816-820 lie at approximately the same positions on the distance axisfrom one assay to the next when the electrophoresis conditions areconstant, as they are in apparatus 30, any peaks that lie outside spikes816, 818, and 820 can be eliminated as spurious. For example, the smallpeak shown between arrows 824 and 826 may be due to dust or some othercause such as macro creatine kinase, but it is definitely notattributable to the MM, MB or BB isoenzyme of creatine kinase. Suchout-of-position peaks are eliminated during editing step 814.

Furthermore, during editing step 814 a baseline 832 which passes throughthe minima identified by arrows 822-830 is also calculated. The areabeneath baseline 832 may, for example, represent a spurious signal dueto a modified form of albumin present in the patient's blood. Baseline832 is subtracted from spikes 816, 818, and 820 during editing step 814.

While background noise due to modified forms of albumin can be editedelectronically by determining a baseline as noted above, the problem canbe eliminated chemically as an alternative. Methyl red, a Ph indicatordye, binds tightly with albumin and displaces whatever substances maypreviously have been bound to it. Albumin bound to methyl red does notfluoresce and in fact absorbs ultraviolet light. Accordingly, backgroundnoise due to modified albumin can be avoided by adding one percent byvolume of methyl red to the patient's serum and waiting five minutes forit to bind before beginning the electrophoresis procedure. It isbelieved that a reduction in albumin-origin noise could also be achievedby including the Ph indicator dye in the electrophoresis medium layer ofthe electrophoresis plate or in the reagent. Methyl orange can also beused, but superior results are obtained with methyl red.

The six edited scans are displayed sequentially on video monitor 34 (seeFIG. 1) in step 834 and printed by printer 36 in step 836. FIG. 23illustrates the video display and hard copy corresponding to theun-edited scan depicted in FIG. 22 if the option to have the resultsexpressed in international units is elected and if 50 internationalunits are selected to represent full scale. FIG. 24 illustrates the sameinformation if the option to have the results automatically scaled iselected. In both cases, quantitative measures of the relativepercentages of the three fractions and the international units arepreferably also depicted, as illustrated.

Finally, in step 838 gantry assembly 56 is returned to its originalposition in preparation for the next run.

While the program of FIGS. 21A-21M has been described in the context ofan assay of creatine kinase, apparatus 30 can assay other substances,such as lactate dehydrogenase. A lactate dehydrogenase assay is usefulto physicians when diagnosing heart or kidney ailments.

Electrophoresis plate 110 (FIG. 4) is typically packaged with aprotective plastic film (not illustrated) on central portion 120. Due toa phenomenon known as syneresis, liquid is expressed from theelectrophoresis medium layer and accumulates under the protective film.As was noted previously, the electrophoresis medium layer preferablyincludes a surfactant such a methyl cellulose, which alters the surfacetension of the electrophoresis medium layer and causes the accumulatedliquid to wet central portion 120 in a uniform film when the protectiveplastic film is removed prior to use of electrophoresis plate 110.Otherwise, the expressed liquid remaining on central portion 120 wouldform thin, irregular patches when the protective plastic film is removedand these irregular patches would have to be removed prior to use ofplate 110 in order to keep them from undermining the electricalhomogeneity of the electrophoresis medium layer. Typically the operatorremoves irregular patches of liquid from an electrophoresis plate thatlacks a surfactant such as methyl cellulose by blowing them away, forexample, or by blotting the electrophoresis plate.

If electrophoresis plates 110 without methyl cellulose are used withelectrophoresis apparatus 30, the program illustrated in FIGS. 21A-21Mcan readily be modified to remove the irregular patches of liquidautomatically in a preliminary step. This is accomplished using the airknife, with blower 340 operating at high speed as gantry assembly 56sweeps back and forth over electrophoresis plate 110.

It will be apparent that information about various aspects ofelectrophoresis apparatus 30 is needed by computer 62 during executionof the program shown in FIGS. 21A-21M. Some of this information is veryaccurately known at the time apparatus 30 is made. For example, due tomechanical considerations the distance moved by gantry assembly 56during successive pulses from encoder 366 (see FIG. 19) is known withprecision and can be stored in hard disk 504 (see FIG. 17) duringmanufacture of apparatus 30 for use throughout the life of apparatus 30.Other values needed during execution of the program are not preciselyknown during manufacture due to variations in individual components anddue to manufacturing tolerances when electrophoresis apparatus 30 isconstructed. For example, the performance of commercially availabletemperature sensors may vary slightly from one sensor to the next, andthe exact position of slit 282 (see FIG. 16) when it is over apredetermined track 388, in terms of encoder pulses from home switch396, depends upon precisely how the relative components are mounted inelectrophoresis apparatus 30 and may vary slightly from one apparatus 30to the next. Approximately correct default values for such parametersare stored in hard disk 504 when apparatus 30 is made, and it isdesirable to calibrate apparatus 30 prior to use to replace thesedefault values with more accurate values. Calibration procedures will bedescribed below. Another class of information stored in hard disk 504need not be known with extreme accuracy (e.g., the exact position ofelectrophoresis platform 48 in terms of encoder pulses from home switch392 when the reagent is poured at station 54) or can be accuratelydetermined from the calibrated values (e.g., the position ofelectrophoresis platform 48 during the electrophoresis procedure, interms of counted encoder pulses from home switch 392).

FIGS. 25A-25C illustrate the procedure for calibrating platformtemperature sensor 192. An accurate electronic thermometer (notillustrated) with a probe is used during, this procedure. Before theprocedure begins, the probe is inserted on top of heat-transfer member140 (FIG. 6).

Temperature sensor 192 is highly linear and its performance can berepresented very accurately by the following linear Equation (4):

    T=mS+b                                                     (4)

In the above equation, S represents the sensor output in millivolts. Trepresents the temperature. The term m represents the slope of thelinear relationship, and will be called the "resolution." The term brepresents the intercept with the ordinate axis, and will be called the"offset." In the calibration procedure described below the sensor outputis measured at two different temperatures, yielding two linear equations(in the form of Equation 4) which can be solved for the resolution m andthe offset b.

In step 834, default values for the resolution m and offset b are readfrom a sensor calibration register. These values are available from themanufacturer of the sensor or can easily be determined from other dataprovided by the manufacturer. Using the default values, the sensoroutput is calculated for a temperature of 10° C. using Equation 4. Thecalculated sensor output at 10° C. will be designated S_(LOW). Thecalculated sensor output S_(LOW) is stored in step 838, and in step 840Peltier power supply 514 (see FIG. 19) drives Peltier devices 186 sothat the output of platform temperature sensor 192 becomes S_(LOW).Power supply 514 controls Peltier devices 186 in a closed loop servocontrol; Peltier devices 186 are driven until sensor 192 outputs thedesired output and then the drive current is reduced until the output ofsensor 192 departs slightly from the desired output, whereupon Peltierdevices 186 are driven with more current. In this way the temperature iscontrolled by hardware within a narrow band.

The present temperature sensed by sensor 192 is calculated in step 842using Equation 4 and the default resolution and offset. This isdisplayed on monitor 34 (see FIG. 1) in step 844. In step 846 a check ismade to determine whether the calculated present temperature has reached10° C. yet. After it has, the operator is asked to enter the measuredtemperature in step 848. Here, the measured temperature refers to thetemperature sensed by the electronic thermometer. After the measuredtemperature has been entered (step 850), it is stored as T_(LOW) in step852. Next, a sensor output S_(HIGH) is calculated for a temperature of55° C. in step 854. The calculated output S_(HIGH) is stored in step856, and in step 858 Peltier devices 186 are driven by the hardware toachieve S_(HIGH) as an output from sensor 192. The present temperatureis calculated on the basis of the Equation 4 and the present output ofsensor 192 in step 860, and this calculated temperature is displayed instep 862. A check is made in step 864 to determine whether thecalculated temperature has reached 55° C. yet. When it has, the operatoris asked in step 866 to enter the temperature measured using theelectronic thermometer. After he has entered the measured temperature,step 868, it is stored as T_(HIGH) in step 870. At this point twomeasured values of the temperature (T_(LOW) and T_(HIGH)) andcorresponding sensor outputs (S_(LOW) and S_(HIGH)) are available, sothe actual offset b and resolution m can be calculated in step 872. Theactual values are then stored in the sensor calibration memory (step874) in lieu of the previously-stored default resolution and offset.

Gantry temperature sensor 443 (see FIG. 19) is calibrated in the sameway. The temperature probe of the electronic thermometer (notillustrated) is placed at air knife slot 338 and blower 340 is turned onduring the calibration procedure. In this case, the low temperatureselected for the calibration procedure is 35° C. and the hightemperature is 63° C.

A similar procedure is employed to calibrate electrophoresis powersupply 64. Instead of a sensor output signal, the variable in the linearequation is a command value from computer 62 which is used with theoffset and resolution to determine a control signal for power supply 64.Initially, a voltmeter (not illustrated) is placed across the powersupply. Default values for the voltage resolution and voltage offset areemployed with a command value for a relatively low voltage (200 volts)and a command signal for a relatively high voltage (1200 volts) tocompute low voltage and high voltage control signals for power supply64. The measured values for the voltage can then be used to find theactual offset and resolution for use with any command values.

The current response of power supply 64 is calibrated in a similarmanner. A milliammeter is connected across the output of power supply 64in series with a 5490 ohm load resistor. Default values for the currentresolution and current offset are then used to generate control valuescommanding a 20 milliamp output and a 91 milliamp output. The actualvalues obtained from the milliammeter can then be used, in conjunctionwith the control values supplied by computer 64 for calculating thecontrol signal, to calculate the actual offset and resolution.

The procedure for calibrating electrophoresis platform 48 and gantryassembly 56 will now be described with reference to FIGS. 16, 20, and 26and the flow chart shown in FIGS. 27A-27D. The purpose in thiscalibration procedure is not to determine actual offset and resolutionvalues for use with a linear equation, but instead to determine thetotal number of encoder pulses from home switches 392 and 396 whenplatform 48 and gantry assembly 56 are in predetermined positions.

A calibration template 876 is used when platform 48 and gantry assembly56 are calibrated. It is a thin rectangular plate made of hard plastic,and has a hole 878, a slot 880, and rectangular openings 882 and 884through it. Before the calibration procedure, template 876 is installedin recessed region 132 (see FIG. 5) of tray 130, with alignment peg 150extending through hole 878 and with alignment peg 148 extending throughslot 880. This precisely positions template 876 with respect toelectrophoresis platform 48. Electrodes 146 extend through opening 882in template 876 and electrodes 144 extend through opening 884.

The upper surface of template 876 is black, and absorbs ultravioletradiation. Fluorescent alignment marks are provided on the blacksurface. These include six pipette alignment dots 886 which, as will bediscussed, are used to determine the precise position of platform 48when it is beneath pipettes 52, in terms of the total number of encoderpulses from home switch 392. The alignment marks additionally include agantry alignment line 888. It is used to find the exact relationship ofplatform 48 with respect to slit 282, again in terms of the total numberof encoder pulses from home switch 392. Gantry alignment line 888 isparallel to slit 282, and its vertical position (with respect toalignment hole 878, FIG. 26) on template 76 is known. Furthermore thedistance moved by electrophoresis platform 48 between two pulses ofposition encoder 372 is known (such as one thousandth of an inch perpulse). By scanning the line 888, that is, by moving platform 48 alongplatform path 390 (FIG. 16) until slit 282 lies directly over line 888and fluorescent light emitted by line 888 is detected by photomultipliertube 312, the location of alignment pin 150 (FIG. 5) can be determined.All positions along platform path 390 for scanning, sample application,etc., are referenced to pin 150. Finally, the adjacent marks include sixtrack alignment lines 890-900, one for each of the six tracks 388 (FIG.16). They are used to determine the exact position of each track 388 interms of the total number of pulses of encoder 366 when gantry assembly56 moves from home switch 396 to a position in which slit 282 isdirectly above the respective track 388.

Turning now to the flow chart shown in FIGS. 27A-27D, a check is made instep 902 to determine whether gantry assembly 56 is located at homeswitch 396. If not, in step 904 it is moved to home switch 396 and agantry position counter is cleared in step 906. A check is then made instep 908 to determine whether electrophoresis platform 48 is positionedat its home switch 392, and if not it is moved to that position in step910. A platform position counter is cleared in step 912. Then a defaultposition for applicator assembly 50 is loaded into an applicatorposition register in step 914, and platform 48 is moved until it reachesthat position in step 916. Pipettes 52 (see FIG. 2) are then lowered toa position slightly above template 876 (step 918). The exact distance isnot critical.

In step 920 the operator is asked whether pipettes 52 are positionedover pipette alignment dots 886. After the operator has responded (step922), a check is made to determine whether the operator has entered "N,"indicating that pipettes 52 are in front of or behind dots 886 and thatadjustments are needed. If adjustments are needed, in step 926 theoperator is asked to press keys on keyboard 32 to move platform 48backward or forward, as needed. After the operator has complied (step928) the content of applicator position register is decremented ifplatform 48 needs to be moved backward or incremented if it needs to bemoved forward (step 930). Processing then returns to step 916. Whenpipettes 52 finally lie above dots 886, the value in the applicatorposition register is stored as a replacement for the default value instep 932.

Although not shown in detail, applicator assembly 50 is mounted so thatit is laterally adjustable. In step 934 the operator is asked tomechanically adjust applicator assembly 50 if necessary to move thepipettes 52 laterally until they are over dots 886. After the operatorhas complied, step 936, gantry assembly 56 is moved to a gantryalignment position in step 938.

In the gantry alignment position, slit 282 is positioned to pass overgantry alignment line 888 when platform 48 is moved back. Since gantryalignment line 888 is relatively long it will be apparent that thegantry alignment position is not at all critical, and can be set withassurance that slit 828 will indeed encounter alignment line 888 even ifthere are considerable variations from one apparatus 30 to the next dueto manufacturing tolerances. In step 940, electrophoresis platform 48 ismoved towards its home switch 392 until slit 282 encounters thealignment line 888. The counted number of pulses of encoder 372 betweenhome switch 392 and this position is stored in replacement of a defaultvalue that was stored when electrophoresis apparatus 30 was made. Theprogram then proceeds to determining the exact positions of the tracksalong gantry path 394, in terms of counted pulses emitted by encoder 366from home switch 396. In step 942, a track counter is set to one.Platform 48 is moved to a position for sensing track alignment line 890in step 944. This is the approximate position where gantry path 394traverses alignment line 890, and due to the length of line 890 it willbe apparent that this position is not critical. Gantry assembly 56 isthen moved to a gantry start position in step 946. This position, againnot critical, is located at the right (with respect to FIG. 26) of trackalignment lines 890-900. Gantry assembly 56 is then moved to the rightin step 948 until it senses alignment line 898. It will be noted thatthe track alignment lines 890-900 overlap slightly at their ends, and itis desirable for an expected range of positions for each alignment lineto be established in order to avoid the chance that an unintendedalignment line might be detected if a particular apparatus 30 isproduced at the extreme range of manufacturing tolerances. A value forthe counted pulses from encoder 366 when the alignment line is detectedis stored in lieu of a default value that was stored when assembly 30was manufactured.

In step 950 the track counter is incremented, and in step 951 a check ismade to determine whether any more tracks remain to be calibrated. Whenthe sixth track has been calibrated, electrophoresis platform 48 ismoved to the front of apparatus 30 (step 952) and the calibrationprocedure is finished.

The procedure for calibrating applicator assembly 50 will now bedescribed with reference to FIGS. 17, 20, and 29 and the flow chartshown in FIGS. 28A-28D.

In step 953 a check is made to determine whether plate 400 is at its topposition, that is, whether home switch 416 is closed. If not, motor 406is actuated to move plate 400 to its top position in step 954, and thena barrel position counter is cleared in step 955. In step 956 a check ismade to determine whether plungers 956 are at their top position, thatis, whether home switch 436 is closed. If not, motor 430 is actuated instep 957 to move them to their top position and a plunger positioncounter is cleared in step 958. A check is made in step 959 to determinewhether electrophoresis platform 48 is in its rear position, and if notit is moved to the rear position in step 960, whereupon the platformposition counter is cleared in step 961.

Platform 48 is moved beneath applicator assembly 50 in step 962. Moreparticularly, platform 48 is moved so that pipettes 52 lie above thecentral region of protective film 142 (see FIG. 5). A default value thatwas stored when apparatus 30 was manufactured is then loaded into apipette position register in step 963. If it turns out to be accurate,the default value will equal the counted pulses from encoder 408 whenthe lower tips of barrels 422 are located at a position greater than0.027 inches and less than 0.037 inches above film 142. In step 964,motor 406 is actuated to move the pipettes 52 to the position indicatedin the pipette position register. The operator is then asked, in step965, to use a barrel feeler gauge to check the distance between thelower tips of barrels 422 and protective film 142. A barrel feeler gauge966 is shown in FIG. 29. It is a go/no-go gauge having a portion 967with a thickness of 0.027 inches and a portion 968 with a thickness of0.037 inches. The operator checks to see whether portion 967 can slideunderneath barrels 422 and portion 968 cannot. After the operator hasused feeler gauge 966 and entered the result (step 969), a check is madeat step 970 to determine whether adjustment is needed. If so, theoperator is asked to press keys on keyboard 32 (see FIG. 1) to indicatewhether barrels 422 are too high or too low. After he has complied, step972, the value in the pipette position register is incremented ordecremented as appropriate in step 973.

In step 974, motor 406 is actuated to raise plate 400 until home switch916 is closed, and then processing returns to step 964 for furtheradjustment. Step 974 is not preparatory to clearing any counters;instead, the purpose of raising plate 400 is to ensure that calibrationis accomplished by moving the elements in the same direction during asequence of trials and refinements so as to avoid erratic results due tomechanical backlash.

When the distance has finally been adjusted so that portion 967 offeeler gauge 966 slides beneath barrels 422 but portion 968 does not(the "Yes" decision at step 970), the default value stored at the timeof manufacture is replaced by the value in the pipette position counter(step 975). The program then proceeds to calibration of plungers 440 sothat the amount of fluid they draw in or discharge can be determinedwith accuracy.

In step 976, a first plunger default value is loaded into a firstplunger position register. If the default value turns out to beaccurate, it will equal the counted pulses from encoder 431 when thelower ends of plungers 440 are even with the lower ends of barrels 422.This is the zero microliter position. In step 977, motor 430 is actuatedto bring plungers 440 to the position indicated in the first barrelposition register. The operator is then asked to use a first barrelposition gauge to check the distance (step 978). The first barrel feelergauge is a go/no-go gauge with a thick portion and a thin portion, likethe barrel feeler gauge 966 shown in FIG. 29. The first plunger feelergauge is used to determine the distance between bars 418 and 438 (thelengths of plungers 440 are selected such that the lower ends of theplungers would protrude below barrels 422 if bar 438 were moved intocontact with bar 418). After the operator has used the feeler gauge(step 979), a decision is made as to whether adjustment is necessary(step 980). If so, in step 981 the operator is asked to use keyboard 32to indicate whether plungers 440 should be lowered or raised (step 981).After the operator has done this (step 982), the content of the firstplunger position register is incremented or decremented in accordancewith the operator's entry (step 983) and motor 430 is actuated to raiseyoke 424 until home switch 436 is closed (step 984). This is not apreliminary to clearing any position counters but, instead, ensures thatplungers 440 are moved in the same direction during the calibrationprocedure to avoid inconsistent results due to mechanical backlash.After the plungers have been raised, processing returns to step 977.

After plungers 440 have been calibrated at the zero microliter positionusing the first plunger feeler gauge, the value in the first plungerposition register is stored in lieu of the default value (step 985).Then, in step 986, steps 976 through 985 are repeated to calibrate a onemicroliter position using a second barrel feeler gauge, which is againused to determine the distance between bars 418 and 438.

It will be understood that the above description of the presentinvention is susceptible to various modifications, changes, andadaptations, and the same are intended to be comprehended within themeaning and range of equivalence of the appended claims.

What we claim is:
 1. A method for analyzing a liquid specimen,comprising the steps of:(a) depositing the specimen on anelectrophoresis medium layer; (b) establishing an electric field acrossthe electrophoresis medium layer; (c) pouring a reagent on theelectrophoresis medium layer; (d) spreading the reagent by blowing gasagainst the electrophoresis medium layer through an air knife slot whilemoving the air knife slot and the electrophoresis medium layer withrespect to one another; (e) shining ultraviolet light on theelectrophoresis medium layer; and (f) scanning the electrophoresismedium layer with an optical sensor.
 2. A method for assaying isoenzymesof creatine Kinase in a liquid sample, comprising the steps of:(a)placing the liquid sample in a receptacle; (b) transferring the sampleto an electrophoresis medium layer; (c) establishing an electric fieldacross the electrophoresis medium layer; (d) depositing a reagent on theelectrophoresis medium layer; (e) shining ultraviolet light on theelectrophoresis medium layer; (f) scanning the electrophoresis mediumlayer with an optical sensor; and (g) exposing the sample to a Phindicator dye before step (f) is conducted.
 3. The method according toclaim 1 including the step of positioning the electrophoresis mediumlayer on a support for an electrophoresis apparatus, the apparatuscomprising:a platform having electrodes that contact the electrophoresismedium and an optical detector; the method further including the stepsof: moving the electrophoresis support along a first path; moving theoptical detector along a second path that passes over the first path. 4.The method according to claim 3 and further including the stepof:blowing air on the electrophoresis medium.
 5. The method according toclaim 4 and further including the step of heating the air which is blownon the electrophoresis medium.
 6. The method according to claim 1including the step of calibrating the position of the electrophoresismedium layer.